Regulation of transforming growth factor-beta (TGF-β) gene expression in living cells via the application of specific and selective electric and electromagnetic fields

ABSTRACT

Methods and devices are described for the regulation of Transforming Growth actor (TGF)-β1, β2, and/or β3 protein gene expression in bone cells and other tissues via the capacitive coupling or inductive coupling of specific and selective electric fields to the bone cells or other tissues, where the specific and selective electric fields are generated by application of specific and selective electric and electromagnetic signals to electrodes or one or more coils or other field generating device disposed with respect to the bone cells or other tissues so as to facilitate the treatment of diseased or injured bone and other tissues. By gene expression is meant the up-regulation or down-regulation of the process whereby specific portions (genes) of the human genome (DNA) are transcribed into mRNA and subsequently translated into protein. Methods and devices are provided for the targeted treatment of injured or diseased bone and other tissue that include generating specific and selective electric and electromagnetic signals that generate fields in the target tissue optimized for increase of TGF-β1, β2, and/or β3 protein gene expression and exposing bone and other tissue to the fields generated by specific and selective signals so as to regulate TGF-β1, β2, and/or β3 protein gene expression in such tissue. The resulting methods and devices are useful for the targeted treatment of bone fractures, fractures at risk, delayed unions, nonunion of fractures, bone defects, spine fusions, osteonecrosis or avascular necrosis, as an adjunct to other therapies in the treatment of one or all of the above, in the treatment of osteoporosis, and in other conditions in which TGF-β1, β2, and/or β3 protein may be implicated.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application is a continuation-in-part patentapplication of U.S. patent application Ser. No. 10/257,126, filed Oct.8, 2002, which is the U.S. national phase patent application ofPCT/US01/05991, filed Feb. 23, 2001, which, in turn, claims the benefitof the filing date of U.S. Provisional Patent Application Ser. No.60/184,491, filed Feb. 23, 2000.

FIELD OF THE INVENTION

The present invention is directed to a method of up-regulatingtransforming growth factor-beta (TGF-β) gene expression in living cellsvia the application of electric and electromagnetic fields generated byspecific and selective electric and electromagnetic signals for thetreatment of injured or diseased tissues, as well as devices forgenerating such signals.

BACKGROUND OF THE INVENTION

The bioelectrical interactions and activity believed to be present in avariety of biological tissues and cells are one of the least understoodof the physiological processes. However, there has recently been muchresearch into these interactions and activity regarding the growth andrepair of certain tissues and cells. In particular, there has been muchresearch into stimulation by electric and electromagnetic fields and itseffect on the growth and repair of bone, cartilage, and various growthfactors. Researchers believe that such research may be useful in thedevelopment of new treatments for a variety of medical problems.

Transforming growth factor—beta (TGF-β) is a pleiotropic growth factorthat is present in most tissues and is implicated in cell proliferation,migration, differentiation, and survival. Consequently, TGF-β hasclinical applications in diverse conditions such as angiogenesis,autoimmunity, bone repair (fractures, delayed unions, nonunions) andbone maintenance (osteoporosis), cartilage maintenance (degenerativearthritis), tumor suppression, and wound healing (Kim et al., J ofBiochemistry and Molecular Biology, 38: 1-8, (2005); Janssens et al.,Endocrine Reviews, 26: 743-774, (2005).

In acute fractures, delayed union and nonunion of fractures, and invarious defects of bone, the formation of new, healing bone is dependentupon the presence of bone morphogenetic proteins (BMPs) to induce boneformation and TGF-βs to induce cartilage formation. In PCT PatentApplication Serial No. PCT/US2005/00793, filed Jan. 11, 2005 (claimingpriority from U.S. Provisional Patent Application No. 60/535,755, filedJan. 12, 2004), it was shown that the gene expression of BMPs could beup-regulated by specific and selective electric and electromagneticfields for the treatment of injured or diseased bone. It is shown hereinthat the gene expression of TGF-βs can also be up-regulated by specificand selected electric and electromagnetic fields. It is also shownherein that the optimal signal for the gene expression of BMPs isslightly different from that of TGF-βs, and this difference allows oneto design a device that delivers one signal that maximally up-regulatesthe BMPs during the bone phase of fracture healing and another signalthat primarily up-regulates the TGF-βs during the cartilage phase offracture healing. This would be very useful in fracture healing, forinstance, where the fracture callus is initially composed of cartilagethat gradually is replaced by bone. By maximally up-regulating theTGF-βs to form cartilage early in the healing process, and maximallyup-regulating the BMPs to form bone later in the healing process, one isable to optimize the healing of acute fractures, accelerate the healingin delayed fracture healing, and restart the healing process in nonunionfractures.

Up-regulation of TGF-β may also be useful in the treatment of thedisease commonly known as osteoporosis, where bone demineralizes andbecomes abnormally rarefied. Bone comprises an organic component ofcells and matrix as well as an inorganic or mineral component. The cellsand matrix comprise a framework of collagenous fibers that isimpregnated with the mineral component of calcium phosphate (85%) andcalcium carbonate (10%) that imparts rigidity to the bone. In healthybone, bone formation and bone resorption are in balance. Inosteoporosis, bone resorption exceeds bone formation, leading to boneweakening and possible vertebral body fracture and collapse. Whileosteoporosis is generally thought as afflicting the elderly, certaintypes of osteoporosis may affect persons of all ages whose bones are notsubject to functional stress. In such cases, patients may experience asignificant loss of cortical and cancellous bone during prolongedperiods of immobilization. Elderly patients are known to experience boneloss due to disuse when immobilized after fracture of a bone, and suchbone loss may ultimately lead to a secondary fracture in an alreadyosteoporotic skeleton. Diminished bone density may lead not only tovertebrae collapse, but also to fractures of hips, lower arms, wrists,ankles as well as incapacitating pains. Alternative non-surgicaltherapies for such diseases are needed.

Pulsed electromagnetic fields (PEMF) and capacitive coupling (CC) havebeen used widely to treat nonhealing fractures (nonunion) and relatedproblems in bone healing since approval by the Food and DrugAdministration in 1979. The original basis for the trial of this form oftherapy was the observation that physical stress on bone causes theappearance of tiny electric currents that, along with mechanical strain,were thought to be the mechanisms underlying transduction of thephysical stresses into a signal that promotes bone formation. Along withdirect electric field stimulation that was successful in the treatmentof nonunion, noninvasive technologies using PEMF and capacitive coupling(where the electrodes are placed on the skin in the treatment zone) werealso found to be effective. PEMFs generate small, induced currents(Faraday currents) in the highly-conductive extracellular fluid, whilecapacitive coupling directly causes currents in the tissues; both PEMFsand CC thereby mimic endogenous electrical currents.

The endogenous electrical currents, originally thought to be due tophenomena occurring at the surface of crystals in the bone, have beenshown to be due primarily to movement of fluid containing electrolytesin channels of the bone containing organic constituents with fixednegative charges, generating what are called “streaming potentials.”Studies of electrical phenomena in bone have demonstrated amechanical-electrical transduction mechanism that appears when bone ismechanically compressed, causing movement of fluid and electrolytes overthe surface of fixed negative charges in the proteoglycans and collagenin the bone matrix. These streaming potentials serve a purpose in bone,and, along with mechanical strain, lead to signal transduction that iscapable of stimulating bone cell synthesis of a calcifiable matrix, and,hence, the formation of bone.

The main application of direct current, capacitive coupling, and PEMFshas been in orthopedics in healing of nonunion bone fractures (Brightonet al., J. Bone Joint Surg. 63: 2-13, 1981; Brighton and Pollack, J.Bone Joint Surg. 67: 577-585, 1985; Bassett et al., Crit. Rev. Biomed.Eng. 17: 451-529, 1989; Bassett et al., JAMA 247: 623-628, 1982).Clinical responses have been reported in avascular necrosis of hips inadults and Legg-Perthes's disease in children (Bassett et al., Clin.Orthop. 246: 172-176, 1989; Aaron et al., Clin. Orthop. 249: 209-218,1989; Harrison et al., J. Pediatr. Orthop. 4: 579-584, 1984). It hasalso been shown that PEMFs (Mooney, Spine 15: 708-712, 1990) andcapacitive coupling (Goodwin, Brighton et al., Spine 24: 1349-1356,1999) can significantly increase the success rate of lumbar fusions.There are also reports of augmentation of peripheral nerve regenerationand function and promotion of angiogenesis (Bassett, Bioessays 6: 36-42,1987). Patients with persistent rotator cuff tendonitis refractory tosteroid injection and other conventional measures, showed significantbenefit compared with placebo-treated patients (Binder et al., Lancet695-698, 1984). Finally, Brighton et al. have shown in rats the abilityof an appropriate capacitive coupling electric field to both prevent andreverse vertebral osteoporosis in the lumbar spine (Brighton et al., J.Orthop. Res. 6: 676-684, 1988; Brighton et al., J. Bone Joint Surg. 71:228-236, 1989).

More recently, research in this area has focused on the effectsstimulation has on tissues and cells. For example, it has beenconjectured that direct currents do not penetrate cellular membranes,and that control is achieved via extracellular matrix differentiation(Grodzinsky, Crit. Rev. Biomed. Eng. 9:133-199, 1983). In contrast todirect currents, it has been reported that PEMFs can penetrate cellmembranes and either stimulate them or directly affect intracellularorganelles. An examination of the effect of PEMFs on extracellularmatrices and in vivo endochondral ossification found increased synthesisof cartilage molecules and maturation of bone trabeculae (Aaron et al.,J. Bone Miner. Res. 4: 227-233, 1989). More recently, Lorich et al.(Clin. Orthop. Related Res. 350: 246-256, 1998) and Brighton et al. (J.Bone Joint Surg. 83-A, 1514-1523, 2001) reported that signaltransduction of a capacitively coupled electric signal is via voltagegated calcium channels, whereas signal transduction of PEMFs or combinedelectromagnetic fields is via the release of calcium from intracellularstores. In all three types of electrical stimulation there is anincrease in cytosolic calcium with a subsequent increase in activated(cytoskeletal) calmodulin.

It was reported in 1996 by the present inventors that a cyclic biaxial0.17% mechanical strain produces a significant increase in TGF-β₁ mRNAin cultured MC3T3-E1 bone cells in a cooper dish (Brighton et al.,Biochem. Biophys. Res. Commun. 229: 449-453, 1996). Several significantstudies followed in 1997. In one study it was reported that the samecyclic biaxial 0.17% mechanical strain produced a significant increasein PDGF-A mRNA in similar bone cells (Brighton et al., Biochem. Biophys.Res. Commun. 43: 339-346, 1997). It was also reported that a 60 kHzcapacitively coupled electric field of 20 mV/cm produced a significantincrease in TGF-β₁ in similar bone cells in a cooper dish (Brighton etal., Biochem. Biophys. Res. Commun. 237: 225-229, 1997). However, theeffect such a field would have on other genes within the body has notbeen reported in the literature.

In the above-referenced parent patent application, entitled Regulationof Genes Via Application of Specific and Selective Electrical andElectromagnetic Signals, methods were disclosed for determining thespecific and selective electrical and electromagnetic signals for use increating fields for regulating target genes of diseased or injuredtissues. The present invention builds upon the technique describedtherein by describing the method of regulating expression of onetargeted gene family, namely, TGF-β's gene expression, throughapplication of a field generated by a specific and selective electricaland electromagnetic signal, for the treatment of fresh fractures,fractures at risk, delayed unions, nonunion of fractures, bone defects,spine fusions, osteonecrosis or avascular necrosis, as an adjunct toother therapies in the treatment of one or all of the above, and in thetreatment of osteoporosis.

SUMMARY OF THE INVENTION

The present invention relates to regulating transforming growthfactor-beta (TGF-β) gene expression in bone cells (as an example) viathe application of specific and selective electric and/orelectromagnetic fields generated by specific and selective electricand/or electromagnetic signals applied to electrodes. By performingsequential dose-response curves on the electric signal duration,amplitude, frequency, and duty cycle in which the effects of theresultant electric field are measured, the optimal signal forup-regulating TGF-β mRNA in bone cells was discovered. The optimalsignal generated a capacitively coupled electric field with an amplitudeof 20-40 mV/cm, a duration of 24 hours, a frequency of 60 kHz, and aduty cycle of 50%. In particular, the present invention relates toup-regulating TGF-β 1, 2, and 3 gene expression in bone cells via theapplication of fields generated by such signals.

In an exemplary embodiment of the invention, methods are provided tospecifically and selectively up-regulate the gene expression (asmeasured by mRNA) of TGF-β1, TGF-β2, and TGF-β3 with capacitivelycoupled electric fields, electromagnetic fields, or combined fields.Fresh fractures, fractures at risk, delayed unions, nonunion fractures,bone defects, osteonecrosis, osteoporosis, and the like are treated witha capacitively coupled electric field of about 20-40 mV/cm with anelectric field duration of about 24 hours, a frequency of 60 kHz, a dutycycle of about 50%, and a sine wave configuration that causes theexpression of TGF-βs 1, 2, and 3 to be up-regulated. In accordance withthe method of the invention, a “specific and selective” signal is asignal that has predetermined characteristics of amplitude, duration,duty-cycle, frequency, and waveform that up-regulates the expression ofthe TGF-β genes (specificity). This allows one to choose differentsignals to up-regulate TGF-β gene expressions in order to achieve agiven biological or therapeutic response (selectivity). The inventionfurther relates to devices employing the methods described herein togenerate specific and selective signals that create electric fields toup-regulate the expression of TGF-β genes.

In related aspects, the invention relates to methods and devices for thetreatment of fresh fractures, fractures at risk, delayed unions,nonunions, bone defects, spine fusion, osteonecrosis, as an adjunct toother therapies treating one or more of the above, and in the treatmentof osteoporosis. The method of the invention also includes themethodology for determining the “specific and selective” signal forTGF-β gene expression by methodically varying the duration of a startingsignal known to increase, or suspected to increase, cellular productionof TGF-βs. After finding the optimal duration, the amplitude of thesignal is varied for the optimal duration of time as determined by thegene expression of TGF-β 1, 2, and 3. The duty cycle, frequency, andwaveform are varied methodically in the same dose response manner asabove while keeping the other signal characteristics constant. Thisprocess is repeated until the optimal signal is determined that producesthe greatest increase in the expression of TGF-βs.

These and other aspects of the present invention will be elucidated inthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the mRNA expression of TGF-βs 1,2, and 3 when bone cells are exposed to a 20 mV/cm capacitively coupledelectric field for various time durations. As indicated, the maximumexpression for the various TGF-β mRNAs occurred with a signal of 24hours duration.

FIG. 2 is a graphic representation of the mRNA expression of TGF-β 1, 2,and 3 when bone cells are exposed to various capacitively coupledelectric field amplitudes with a duration of 24 hours. As indicated, themaximum expression for the various TGF-β mRNAs occurred with a fieldamplitude of 20-40 mV/cm.

FIG. 3 is a graphic representation of the mRNA expression of TGF-β 1, 2,and 3 when bone cells are exposed to various capacitively coupledelectric field frequencies with a field amplitude of 20-40 mV/cm and asignal duration of 24 hours. As indicated, the maximum expression forthe various TGF-β mRNAs occurred with a frequency of 60 kHz.

FIG. 4 is a graphic representation of the mRNA expression of TGF-β 1, 2,and 3 when bone cells are exposed to various capacitively coupledelectric field duty cycles with a frequency of 60 kHz, a field amplitudeof 20 mV/cm, and a signal duration of 24 hours. As indicated, themaximum expression for the various TGF-β mRNAs occurred with a 50% to100% duty cycle with a sine wave configuration.

FIG. 5 is a graphic representation of the TGF-β1 protein when bone cellsare exposed 24 hours to a capacitively coupled electric field of a 50%duty cycle with a field amplitude of 20 mV/cm or 40 mV/cm, a frequencyof 60 kHz, and a sine wave configuration. As indicated, the amount ofTGF-β1 protein increase was the same with either 20 or 40 mV/cm.

FIG. 6 is a graphic representation of BMP-2 protein when bone cells areexposed 24 hours to a capacitively coupled electric field of a 50% dutycycle with a field amplitude of 20 mV/cm or 40 mV/cm, a frequency of 60kHz, and a sine wave configuration. As indicated, unlike the TGF-β1response shown in FIG. 5, there was no significant increase in BMP-2protein production at a field of 40 mV/cm as compared to that occurringat 20 mV/cm.

FIG. 7 is a graphic representation of BMP mRNA expression when bonecells are exposed to a 50% duty cycle, capacitively coupled electricfield (20 mV/cm, 60 kHz, sine wave) for 24 hours. Comparing this figureto FIG. 2 shows the clear distinction between the lack of response ofthe bone cell production of BMP protein in a 40 mV/cm field versus thevery significant increase of the bone cell production of TGF-β proteinin a 40 mV/cm field.

FIG. 8 illustrates the BMP gene expression of FIG. 7 on the same graphas the TGF-β gene expression of FIG. 2.

FIG. 9 is a diagram illustrating a device for the treatment ofosteoarthritis of the knee, in accordance with a preferred embodiment ofthe present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The invention will be described in detail below with reference to FIGS.1-9 Those skilled in the art will appreciate that the description givenherein with respect to those figures is for exemplary purposes only andis not intended in any way to limit the scope of the invention. Allquestions regarding the scope of the invention may be resolved byreferring to the appended claims.

The present invention is based on the discovery that the expression ofcertain genes can be regulated by the application of fields generated byspecific and selective electric and/or electromagnetic signals. In otherwords, it has been discovered by the present inventor that there is aspecific electric and/or electromagnetic signal that generates a fieldfor regulating each gene in bone, cartilage and other tissue cells andthat these specific signals are capable of specifically and selectivelyregulating the genes in such cells. In particular, gene expressiongoverning the growth, maintenance, repair, and degeneration ordeterioration of tissues or cells can be regulated in accordance withthe invention via the application of fields generated by specific andselective electric and/or electromagnetic signals so as to produce asalutary clinical effect. Such discoveries are useful in the developmentof treatment methods that target certain medical conditions includingfresh bone fractures, fractures at risk, delayed union, nonunion, bonedefects, spine fusion, osteonecrosis, as an adjunct in the treatment ofany one or more of the above, and in the treatment of osteoporosis.

As used herein, the phrase “signal” is used to refer to a variety ofsignals including mechanical signals, ultrasound signals,electromagnetic signals and electric signals output by a device. It isto be understood that the term “field” as used herein refers to anelectrical field within targeted tissue, whether it is a combined fieldor a pulsed electromagnetic field or generated by direct current,capacitive coupling or inductive coupling.

The phrase “remote” is used to mean acting, acted on or controlled froma distance. “Remote” regulation refers to controlling the expression ofa gene from a distance. To provide “remotely” refers to providing from adistance. For example, providing a specific and selective signal from aremote source can refer to providing the signal from a source at adistance from a tissue or a cell, or from a source outside of orexternal to the body.

The phrase “specific and selective” signal means a signal that producesan electric field that has predetermined characteristics of amplitude,duration, duty cycle, frequency, and waveform that up-regulate ordown-regulate a targeted gene or targeted functionally complementarygenes (specificity). This allows one to choose different “specific andselective” signals to up-regulate or down-regulate expression of variousgenes in order to achieve a given biological or therapeutic response(selectivity).

The term “regulate” means to control gene expression. Regulate isunderstood to include both up-regulate and down-regulate. Up-regulatemeans to increase expression of a gene, while down-regulate means toinhibit or prevent expression of a gene.

“Functionally complementary” refers to two or more genes whoseexpressions are complementary or synergistic in a given cell or tissue.

“Tissue” refers to an aggregate of cells together with theirextracellular substances that form one of the structural materials of apatient. As used herein, the term “tissue” is intended to include anytissue of the body including muscle and organ tissue, tumor tissue aswell as bone or cartilage tissue. Also, the term “tissue” as used hereinmay also refer to an individual cell.

“Patient” refers to an animal, preferably a mammal, more preferably ahuman.

The present invention provides treatment methods and devices that targetcertain tissues, cells or diseases. In particular, the gene expressionassociated with the repair process in injured or diseased tissues orcells can be regulated by the application of fields generated byelectric signals that are specific and selective for the genes to beregulated in the target tissues or cells. Gene expression can beup-regulated or down-regulated by the application of signals that arespecific and selective for each gene or each set of complementary genesso as to produce a beneficial clinical effect. For example, a particularspecific and selective signal may create an electric field thatup-regulates a certain desirable gene expression, while the same oranother particular specific and selective signal may create an electricfield that down-regulates a certain undesirable gene expression. Acertain gene may be up-regulated by a field generated by one particularspecific and selective signal and down-regulated by a field generated byanother specific and selective signal. Those skilled in the art willunderstand that certain diseased or injured tissues can be targeted fortreatment by regulating those genes governing the growth, maintenance,repair, and degeneration or deterioration of the tissues.

The methods and devices of the present invention are based onidentifying those signals that generate fields that are specific andselective for the gene expression associated with certain targeteddiseased or injured tissue. For example, electricity in its variousforms (e.g., capacitive coupling, inductive coupling, combined fields)can specifically and selectively regulate gene expression in targetedtissues or cells in a patient's body by varying the frequency,amplitude, waveform or duty cycle of the applied field for each selectedgene. The duration of time exposed to electricity can also influence thecapability of electricity to specifically and selectively regulate geneexpression in targeted tissues or cells in a patient's body. Specificand selective signals may generate electric fields for application toeach gene systematically until the proper combination of frequency,amplitude, waveform, duty cycle, and duration is found that provides thedesired effect on gene expression.

It is to be understood that a variety of diseased or injured tissues ordisease states can be targeted for treatment because the specificity andselectivity of an electric field for a certain gene expression can beinfluenced by several factors. In particular, an electrical field ofappropriate frequency, amplitude, waveform and/or duty cycle can bespecific and selective for the expression of certain genes and thusprovide for targeted treatments. Temporal factors (e.g., duration oftime exposed to the electrical field) can also influence the specificityand selectivity of an electric field for a particular gene expression.The regulation of gene expression may be more effective (or madepossible) via the application of an electrical field for a particularduration of time. Therefore, those skilled in the art will understandthat the present invention provides for varying the frequency,amplitude, waveform, duty cycle and/or duration of application of anelectric field until the electric field is found to be specific andselective for certain gene expressions in order to provide fortreatments targeting a variety of diseased or injured tissue ordiseases.

Thus, the present invention can provide for targeted treatments becauseit is possible to regulate expression of certain genes associated with aparticular diseased or injured tissue via the application of electricfields generated by specific and selective signals of appropriatefrequency, amplitude, waveform and/or duty cycle for an appropriateduration of time. The specificity and selectivity of a signal generatingan electrical field may thus be influenced so as to regulate theexpression of certain genes in order to target certain diseased orinjured tissue or disease states for treatment. In particular, thepresent invention provides for the targeted treatment of fresh bonefractures, fractures at risk, nonunion, bone defects, spine fusion,osteonecrosis, as an adjunct in the treatment of one or any of theabove, and in the treatment of osteoporosis.

The devices of the present invention are capable of applying a fieldgenerated by specific and selective signals directly to diseased orinjured tissue and/or to the skin of a patient. The devices of thepresent invention may also provide for the remote application ofspecific and selective fields (e.g., application of a field at adistance from diseased or injured tissue yet which yields the desiredeffect within the targeted cells), although it will be appreciated thatcapacitively coupled devices must touch the subject's skin. The devicesof the present invention may include means for attaching the electrodesto the body of a patient in the vicinity of injured or diseased tissuein the case of capacitive coupling. For example, self-adherentconductive electrodes may be attached to the skin of the patient on bothsides of a fractured bone. As shown in FIG. 9, the device 10 of thepresent invention may include self-adherent electrodes 12 for attachingthe device to the body of a patient. For example, the device 10 of thepresent invention may include electrodes attached to a power unit 14that has a VELCRO® patch 16 on the reverse side such that the power unit14 can be attached to a VELCRO® strap (not shown) fitted around a caston the patient. In the case of inductive coupling, the device of thepresent invention may include coils attached to a power unit in place ofelectrodes.

The device 10 of the present invention can be employed in a variety ofways. The device 10 may be portable or may be temporarily or permanentlyattached to a patient's body. The device 10 of the present invention ispreferably non-invasive. For example, the device 10 of the presentinvention may be applied to the skin of a patient by application ofelectrodes adapted for contact with the skin of a patient for theapplication of electric fields generated by the predetermined specificand selective electric signals. Such signals may also be applied viacoils in which time varying currents flow, thus producing specific andselective electromagnetic fields that penetrate the tissue and createthe specific and selective electric fields in the target tissue. Thedevice 10 of the present invention may also be capable of implantationin a patient, including implantation under the skin of a patient.

Those skilled in the art will further understand that the devices of thepresent invention can be provided in a variety of forms including acapacitively coupled power unit with programmed, multiple, switchable,specific and selective signals for application to one pair or tomultiple pairs of electrodes, electromagnetic coils or a solenoidattached to a power unit with switchable, multiple, specific andselective signals, and an ultrasound stimulator with a power supply forgenerating specific and selective signals. Generally speaking, devicepreference is based on patient acceptance and patient compliance. Thesmallest and most portable unit available in the art at the present timeis a capacitive coupling unit; however, patients with extremelysensitive skin may prefer to use inductive coupling units. On the otherhand, ultrasound units require the most patient cooperation, but may bedesirable for use by certain patients.

EXAMPLE

The invention is demonstrated in the following example, which is forpurposes of illustration and is not intended to limit the scope of thepresent invention.

Materials and Methods

MC3T3-E1 bone cells (5×10⁵ cells/cm²) were plated ontospecially-modified Cooper dishes. The cells were grown for seven dayswith the medium changed just prior to beginning of the experimentalcondition. The experimental cell cultures throughout these studies weresubjected to a capacitively coupled 60 kHz sine wave signal electricfield with an output of 44.81 V peak-to-peak. This produced acalculated-field strength in the culture medium in the dishes of 20mV/cm with a current density of 300 μA/cm². Control cell culture disheswere identical to those of the stimulated dishes except that theelectrodes were not connected to a function generator.

At the end of the experiment, total RNA was isolated using TRIzol,according to the manufacturer's instructions, and reversed transcription(RT) using SuperScript II reverse transcriptase was performed.Oligonucleotide primers to be used in the real time RT-PCR techniquewere selected from published cDNA sequences or designed using the PrimerExpress software program. Quantitative real-time analysis of RT-PCRproducts was performed using an ABI Prism® 7000 Sequence DetectionSystem.

The optimal signal for the desired up-regulation of (TGF)genes—including genes for TGF-β1, TGF-β2, TGF-β3, among others—was foundsystematically as follows. An electrical signal known to cause creationof an electric field that increases (or even just suspected to increase)cellular production of a given protein is taken as the starting signalfor determining the specific signal for generating the electric fieldfor the gene expression (mRNA) of that protein. A dose-response curve isfirst performed by varying the duration of the signal while holding allthe other signal characteristics constant (amplitude, duty cycle,frequency, and waveform) (FIG. 1). This determines the optimal durationof the starting signal for the gene expression of that protein. A seconddose-response curve is then performed, this time varying the amplitudeof the electric field (in mV/cm) while holding the optimal duration andother signal characteristics constant (FIG. 2). A third dose response isperformed, this time varying the signal frequency while holding constantthe optimal duration and optimal amplitude as found previously (FIG. 3).A fourth dose-response is performed varying the duty cycle from 100%(constant) to 10% or less while holding constant the optimal duration,amplitude, and frequency as found previously (FIG. 4). By this method,an optimal signal is determined for producing the greatest increase inthe gene expression of each of the various TGF-betas.

A fifth experiment is performed using a continuous 50% duty cycle(capacitive coupling, 60 kHz, sine wave) to compare a 20 mV/cm field toa 40 mV/cm field in the production of the TGF-β1 protein. As indicated,the TGF-β1 protein increased significantly and equally in the twofields. (FIG. 5). A sixth experiment is performed to demonstrate anincrease in production of the BMP-2 protein in the same two fields asdescribed in FIG. 5 (20 mV/cm and 40 mV/cm). After 24 hours ofstimulation with a 50% duty cycle (capacitively coupled, 60 kHz, sinewave) field, there was a significant increase in BMP-2 protein in the 20mV/cm field but no significant increase in the BMP-2 protein in the 40mV/cm field (FIG. 6.) Thus, and this is very important, one can separatethe expressions of the TGF-β genes from the BMP genes by stimulatingcells with a 40 m v/cm field, even though the genes all belong to thesame TGF super family. This is clearly shown when one compares FIG. 2with FIG. 7. In FIG. 7, a field of 20 mV/cm clearly has a much greaterresponse in up-regulating BMP gene expression than any other field. Bycontrast, in FIG. 2 it is shown that 20 mV/cm and 40 mV/cm fields areequally effective in up-regulating TGF-β gene expression. FIG. 8illustrates the BMP gene expression of FIG. 7 on the same graph as theTGF-β gene expression of FIG. 2. Thus, in fracture healing, one has theoption of stimulating bone and cartilage formation together during theosteogenetic phase of fracture healing or of cartilage only during thecartilage phase of fracture healing.

The present invention clearly shows that the optimal electric fielddescribed in the example can very significantly up-regulate TGF-β 1,2,and 3 mRNA and, hence, increase bone formation in fracture healing,delayed healing, nonunion, bone defects, spine fusions, and inosteoporosis. Those skilled in the art will appreciate that anappropriate electric field, as described herein with capacitivecoupling, is also equally effective with inductive coupling and allelectromagnetic systems that produce equivalent, or nearly equivalent,electric field characteristics. Those skilled in the art will alsoappreciate that more unique signal characteristics may be discoveredthrough more experimentation with more data points (e.g., a 100±3% dutycycle for 30±3 min), but such relatively minor variations in each of thesignal characteristics are believed to be within the level of thoseskilled in the art given the teachings herein.

Those skilled in the art will also appreciate that numerous othermodifications to the invention are possible within the scope of theinvention. For example, the optimal field described herein can beapplied to any bone via two or more appropriate surface electrodes, inpairs or strips, incorporated in braces, wraps, or casts, and deliveredby means of capacitive coupling. Also, the optimal field described herecan be applied to any bone via coil(s) or solenoid incorporated intobraces, wraps, or casts, and delivered by means of inductive coupling.Accordingly, the scope of the invention is not intended to be limited tothe preferred embodiment described above, but only by the appendedclaims.

1. A method of up-regulating the gene expression of Transforming Growth Factor (TGF)-β1, β2, and/or β3 protein in targeted tissue, comprising the steps of: a. generating at least one specific and selective signal having a frequency from 30 kHz to 120 kHz that when applied to a field generating device operatively disposed with respect to said targeted tissue causes the generation of an electric field having an amplitude of about 0.2 to 80 mV/cm in the targeted tissue that is specific and selective for the up-regulation of the gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as measured by mRNA when said electric field is applied to the targeted tissue containing said TGF-β1, β2, and/or β3 protein; and b. exposing said targeted tissue to the specific and selective electric field generated by said field generating device upon application of said at least one specific and selective signal thereto for a predetermined duration of time from approximately ½ hour to 24 hours per 24 hour period at a predetermined duty cycle from approximately 10%-100% so as to selectively up-regulate the gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as measured by mRNA.
 2. The method of claim 1 wherein the generating step comprises the step of selectively varying the amplitude, duration, duty cycle, frequency, and waveform of the applied specific and selective signal until the gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as a result of exposure to the resultant specific and selective electric field as measured by mRNA in the targeted tissue is substantially increased.
 3. The method of claim 1 wherein the exposing step comprises the step of exposing bone cells to the specific and selective electric field for a duration of approximately 24 hours every 24 hours.
 4. The method of claim 1 wherein the generating step comprises the step of generating an electric signal having a sine wave configuration, a duty cycle of approximately 50%, and a frequency of approximately 60 kHz and the resultant specific and selective electric field in the targeted tissue has an amplitude of approximately 20 to 40 mV/cm.
 5. The method of claim 1 wherein said generating step comprises the step of generating the specific and selective signal at a remote source and said exposing step comprises the step of applying the specific and selective electric field to bone tissue.
 6. The method of claim 5 wherein the exposing step comprises the step of applying the specific and selective signal to said field generating device located near the bone tissue.
 7. The method of claim 6 wherein the exposing step comprises the step of applying the specific and selective electric field in the targeted tissue generated by the field generating device upon application of said at least one specific and selective signal thereto to the targeted tissue through capacitive coupling or inductive coupling.
 8. The method of claim 7 wherein when the specific and selective signal is applied to an electrode the electrode generates a capacitive coupling electric field, and when the specific and selective signal is applied to one or more coils said one or more coils generate an electromagnetic field or a combined field.
 9. A method for treating a bone fracture, fracture at risk, delayed union, nonunion, bone defect, spine fusion, osteonecrosis, osteoporosis, and/or other conditions in which Transforming Growth Factor (TGF)-β1, β2, and/or β3 protein has been implicated in a patient, comprising the steps of: a. generating at least one specific and selective signal having a frequency from 30 kHz to 120 kHz that when applied to a field generating device operatively disposed with respect to targeted tissue causes the generation of an electric field having an amplitude of about 0.2 to 80 mV/cm in the targeted tissue that is specific and selective for up-regulating the gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as measured by mRNA when said electric field is applied to the targeted tissue containing said TGF-β1, β2, and/or β3 protein; and b. exposing said targeted tissue to the specific and selective electric field generated by said field generating device upon application of said at least one specific and selective signal thereto for a predetermined duration of time from approximately ½ hour to 24 hours per 24 hour period at a predetermined duty cycle from approximately 10% to 100% so as to selectively up-regulate the gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as measured by mRNA.
 10. The method of claim 9 wherein the exposing step comprises the step of capacitively coupling or inductively coupling the specific and selective electric field to the targeted tissue.
 11. The method of claim 9 wherein the exposing step comprises the step of applying one of an electromagnetic field and a combined field to the targeted tissue.
 12. The method of claim 9 wherein the generating step comprises the step of generating an electric signal having a sine wave configuration, a duty cycle of approximately 50%, and a frequency of approximately 60 kHz and the resultant specific and selective electric field has an amplitude of approximately 20 to 40 mV/cm in the targeted tissue.
 13. The method of claim 9 wherein the exposing step comprises the step of applying the specific and selective electric field to the targeted tissue for a duration of approximately 24 hours every 24 hours.
 14. The method of claim 9 wherein the exposing step comprises the steps of causing the generation of a 20 mV/cm field in the targeted tissue for a selected period of time and causing the generation of a 40 mV/cm field in the targeted tissue for another selected period of time, whereby stimulation of bone morphogenetic protein (BMP) is differentiated from stimulation of (TGF)-β1, β2, and/or β3 protein by the increase in field amplitude from 20 mV/cm to 40 mV/cm.
 15. The method of claim 9 wherein the generating step comprises the steps of starting with any electric signal that when applied to said field generating device generates an electric field that is known or thought to be effective on living cells, performing a first dose-response curve on the duration of stimulation of the electric field to determine an optimal duration; performing a second dose-response curve on the amplitude of the applied electric signal using the optimal duration as previously found to determine an optimal amplitude; performing a third dose-response curve on the frequency of the applied electric signal keeping the optimal duration and optimal amplitude as previously found to determine an optimal frequency; performing a fourth dose-response curve varying the duty cycle of the applied electric signal and keeping the optimal duration, amplitude, and frequency as previously found to determine an optimal duty cycle, and keeping the optimal duration, amplitude, frequency and duty cycle constant while varying the waveform until an optimal waveform for the up-regulation of the gene expression of TGF-β1, β2, and/or β3 protein as measured by mRNA and the indicated protein in the bone tissue is found.
 16. A device for the treatment of a bone fracture, fracture at risk, delayed union, nonunion, bone defect, spine fusion, osteonecrosis, osteoporosis, and/or other conditions in which Transforming Growth Factor (TGF)-β1, β2, and/or β3 protein has been implicated in a patient, comprising a signal source that generates at least one specific and selective signal having a frequency from 30 kHz to 120 kHz and a field generating device connected to the signal source so as to receive said at least one specific and selective signal and that is operatively disposed with respect to targeted tissue, said field generating device upon receipt of said at least one specific and selective signal causing the generation of an electric field having an amplitude of about 0.2 to 80 mV/cm in the targeted tissue that is specific and selective for up-regulating the gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as measured by mRNA, said signal source controlling and varying the duration of time of application of said at least one specific and selective signal for a duration of time from approximately ½ hour to 24 hours per 24 hour period and controlling and varying the duty cycle of said at least one specific and selective signal applied to said field generating device from approximately 10% to 100% so as to selectively up-regulate gene expression of TGF-β1, β2, and/or β3 protein in said targeted tissue as a result of application of said specific and selective electric field thereto.
 17. The device of claim 16 further comprising a portable power unit that drives said signal source in first and second modes, an output signal in the first mode causing the generation of a 20 mV/cm field in the targeted tissue for a selected period of time and an output signal in the second mode causing the generation of a 40 mV/cm field in the targeted tissue for another selected period of time.
 18. The device of claim 17 wherein the portable power unit is programmable such that fields generated during said first and second modes can be sequentially applied to the targeted tissue for various periods of time and in various orders.
 19. The device of claim 16 further comprising means for attaching the field generating device to the body of a patient in the vicinity of bone tissue.
 20. The device of claim 16 further comprising means for attaching the signal source to the body of a patient.
 21. The device of claim 16 wherein the electric field generated by application of said at least one specific and selective signal to the field generating device is applied to said targeted tissue via capacitive coupling or inductive coupling.
 22. The device of claim 21 wherein the specific and selective signal has a sine wave configuration, a duty cycle of approximately 50%, and a frequency of approximately 60 kHz, and the resultant specific and selective electric field has an amplitude of about 20 mV/cm-40 mV/cm in the targeted tissue.
 23. A method of treating a bone fracture, fracture at risk, delayed union, nonunion, bone defect, spine fusion, osteonecrosis, osteoporosis, and/or other conditions in which Transforming Growth Factor (TGF)-β1, β2, and/or β3 protein has been implicated in a patient, comprising the steps of exposing targeted tissue to the specific and selective electric field generated by the device of claim 22 so as to up-regulate gene expression of TGF-β1, β2, and/or β3 protein in the targeted tissue as measured by mRNA in the targeted tissue.
 24. A method of determining a specific and selective electric signal that when applied to a field generating device causes the generation of an electric field in targeted tissue that up-regulates Transforming Growth Factor (TGF)-β1, β2, and/or β3 protein in the targeted tissue, comprising the steps of staffing with a staffing electric signal with a signal shape and frequency that when applied to said field generating device generates an electric field that is known or thought to affect cellular production of TGF-β1, β2, and/or β3 protein, selectively varying a duration of application of said staffing signal until a duration that provides a most significant increase in production of TGF-β1, β2, and/or β3 protein is found, selectively varying an amplitude of the staffing signal until an amplitude that provides a most significant increase in production of TGF-β1, β2, and/or β3 protein is found, selectively varying a duty cycle of the staffing signal until a duty cycle that provides a most significant increase in production of TGF-β1, β2, and/or β3 protein is found, and selectively varying an on-off interval of the duty cycle of the signal until an on-off interval that provides a most significant increase in production of TGF-β1, β2, and/or β3 protein is found.
 25. A method as in claim 24, comprising the further steps of selectively varying a frequency and waveform of said staffing signal, keeping other signal characteristics constant, until a most significant increase in production of TGF-β1, β2, and/or β3 protein as measured by mRNA is found.
 26. The device of claim 16 wherein the field generating device comprises an electrode or one or more coils. 